How insulin binds to and triggers the insulin receptor (IR) represents a fundamental problem at the intersection of basic science and clinical medicine. This application, building on a long-term program of research by a team of investigators, promises to provide a new foundation for analysis of structure-function relationships. Prior progress in the field was limited by the absence of a crystal structure of the hormone-IR complex. In accordance with the Aims of the last renewal, collaborative crystal structures have been obtained of (i) the primary hormone-binding surface of the receptor ectodomain (Site 1; Smith, B.J. et al. Proc. Natl. Acad. Sci. USA 107, 6771-6 (2010)) and (ii) insulin bound to Site 1 (the micro-receptor complex; Menting, J.G. et al. Nature, 493, 241-5 (2013)). The physiological significance of these structures have been validated in the context of the holo-receptor (a) through site-specific photo-cross-linking studies (Xu, B., et al. J. Biol. Chem. 284, 14597-08 (2009)) Paper of the Week and (b) by mutagenesis with application to monogenic syndromes of diabetes mellitus associated with mutations in insulin or its receptor (Whittaker, J., et al. Prc. Natl. Acad. Sci. USA 109, 11166 (2012)). We request continued NIH support to address the following Specific Aims. Aim 1. Functional Mapping of the Functional Surfaces of Insulin. We will employ mutagenesis and residue-specific photo-cross-linking to map the cognate Site-1 and Site-2 binding surfaces of insulin and the IR. Aim 2 Structure of a Model Hormone-Receptor Complex. We seek to improve the resolution and completeness of the micro-receptor structure through synthesis of heavy-atom derivatives of insulin and design of ultra-stable insulin analogs. Inferred structure-function relationships will be tested by non-standard protein design. Aim 3. Biophysical Probes of the Micro-Receptor and Successive Complexes. Two biophysical probes of structure and dynamics will be employed: (a) TROSY-based heteronuclear NMR studies of the micro-receptor complex at 900 MHz and (b) synchrotron-based hydroxyl-radical footprinting (with MS-MS detection) of the micro-receptor and larger receptor fragments, culminating in comparative studies of the holoreceptor. Aim 4. Toward the Structure of a Hormone-Ectodomain Complex. As a long-term goal, we seek to extend our collaboration with M. Lawrence (Melbourne, AU) to a crystal structure of a hormone-ectodomain complex. Together, these Aims represent the culmination of a 25-year program of research that has sought to combine basic science and its potential translation. Insulin and the insulin receptor represent an ancestral signaling system in metazoans of central importance to human health. My colleagues and I are grateful to the Study Section and NIDDK for making this continuing adventure possible.